The purpose of this application is to acquire a confocal microscope system that will be specifically designed for imaging of live cells and embryos under controlled culture conditions. The growing importance of fluorescent protein variants which can act as in vivo reporters for protein expression and subcellular location has accelerated the demand for an instrument that will allow investigators at KUMC to make more use of this technology in live cells. The need to image intracellular calcium fluxes in eggs and embryos have also raised the demand for a system dedicated to live cell imaging. The equipment requested in this proposal has been selected to meet the needs of a group of twelve major users at KUMC and will be operated as a component of the Center for Reproductive Sciences Imaging Core. The request is for a Nikon multiplatform four line confocal microscope that includes a three PMT detector, calcium ratiometric imaging system, image acquisition and analysis software, an enclosed temperature/humidity control system, and dual micromanipulators for microinjection of eggs and single cells. The intended use of this system will be primarily for time lapse observation of live vertebrate cells or embryos for periods of up to 24 hrs, and it will be housed in a separate room dedicated to this purpose. The live cell imaging system will be available to all investigators within the University of Kansas System and to members of surrounding institutions. However, the majority of the research projects using this facility will come from twelve NIH-funded laboratories identified as major users. Dr. William Kinsey will use the instrument for imaging zebrafish and mouse eggs injected with fluorescent calcium reporter molecules. Dr. De Lisle will study secretory vesicle dynamics using GFP constructs. Dr. Godwin will require the ability to image GFP and RFP constructs in living zebrafish embryos. Drs. Hunt and Petroff will use the ratiometric calcium imaging capabilities as well as imaging of GFP constructs. Dr. Little and Dr. Rongish will be doing time-lapse analysis of vascular endothelial or cardiac cells in avian embryos and mammalian embryo explants. Dr. Sarras studies the role of extracellular matrix components in zebrafish development, and will use this facility for time lapse imaging of GFP constructs in live embryos. Dr. Soares will use the facility for microinjection of fluorescent enzyme inhibitors and for calcium imaging in trophoblast cells. Dr. Stephens will image GFP constructs as related to Golgi processing in the Vpu gene in HIV-1 pathogenesis models. Dr. Tash will use the equipment to inject and quantitate fluorescent inhibitors of spermatogenesis. Dr. Terranova will use the equipment to inject and image GFP constructs into mammalian oocytes in the study of the role of Src tyrosine kinase in early development. He will also study calcium transients in granulosa cells induced to divide in vitro with tumor necrosis factor alpha.